Gel-Filtration Chromatography

First, a review of the general principles

of chromatography:
Chromatography literally means “color writing”.

Chromatography was invented by the Russian

botanist Mikhail Tsvet in 1900. He used it to
separate chlorophyll-containing extracts of plants.

Key idea is that molecules of interest interact

differentially with the stationary phase and a mobile
phase, and thus can be separated.
 The Basic Principle
Illustrated

As we discuss
gel-filtration
(aka, size-exlclusion)
chromatography,
compare and
contrast it with ion-
exchange
chromatography

from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005
Partition Coefficient and Relative Mobility

Partition coefficient describes the affinity of a

compound for the stationary phase.
α or (Kav)= molecules adsorbed on stationary phase
molecules in stationary and mobile phase
Can have values between 0 and 1. Example, a
molecule with α = 0.4 will be 40% adsorbed on the
stationary phase.
Relative mobility or retention factor (Rf) describes
the affinity of a molecule for the mobile phase.
Rf = 1 – α (Recall Rf from TLC in Organic Chem)
 Gel-Filtration Chromatography
The principle is somewhat counterintuitive: a
resin is selected that has pores through which
smaller molecules can pass, but larger molecules
are excluded and thus elute first.
The ability of gel filtration to separate molecules
according to size resides with the highly porous
structure of gel filtration media and is basically a
question of accessible volumes.

Scanning electron micrograph of an agarose gel. Magnification x 50,000.

Ref. Anders S. Medin, PhD Thesis, Uppsala University 1995.
Gel-Filtration Chromatography
continued.

from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005
Gel-Filtration Chromatography: another view
 The Role of the Partition Coefficient
In Gel-Filtration Chromatography

where Vt is total volume; Ve is the volume at which the

molecule of interest elutes (assuming that it is within the
fractionation range of the matrix); Vo is the volume of the
space between the beads; and Vi is the volume of the space
within the beads.
see for example, http://www.wiley.com/college/fob/quiz/quiz05/5-6.html
You should learn thoroughly the first equation on the
previous slide (Ve = Vo + KavVi), then you can obtain
the other equations by reminding yourself of the two
extreme values of the partition coefficient Kav.

If Kav = 0 (i.e., if the molecule has no interaction

with the resin and therefore passes around the beads
instead of through the pores of the beads), then
Ve = Vo, and the molecule will come out with the void
volume.

If Kav = 1 (i.e., if the molecule is so small that it has full

access to the pores of the beads), then Ve = Vo + Vi = Vt,
and the molecule will come out with the total volume.
For very small molecules that have full access to the
pores of the beads (small dots), Ve = Vt

excluded
partially
fully
included
included

Fig. 4-4 (Ninfa & Ballou)

 High resolution mode

Gel filtration is
commonly used in two
different modes:

1. High resolution mode

to separate large
molecules like proteins,
peptides
oligonucleotides,
polysaccharides etc.
 Group separation mode
2. Group separation mode to
separate large molecules
(Mr >5000) as a group from
small molecules like salts
and buffer ions.
Group separation mode
accepts much larger sample
volumes than high resolution
mode and can be performed
at considerably higher flow
rates.
 Fractionation Ranges
Matrix name Bead type Approximate
fractionation range for
peptides and globular
proteins (molecular weight)
Sephadex G-50¹ dextran 1500 - 30000
Sephadex G- dextran 4000 - 150000
100¹
Sephacryl S-200 dextran 5000 - 250000
HR¹
Ultrogel AcA 54² polyacrylamide/a 6000 - 70000
garose
Ultrogel AcA 44² polyacrylamide/a 12000 - 130000
garose
Ultrogel AcA 34² polyacrylamide/a 20000 - 400000
garose
Bio-Gel P-60³ polyacrylamide 3000 - 60000
Bio Gel P-150³ polyacrylamide 15000 - 150000
Bio-Gel P-300³
¹Sephadex is a registered trademark of Pharmacia PL.
polyacrylamide 60000 - 400000
²Ultrogel is a registered trademark of Pharmacia-LKB.
³Bio Gel is a registered trademark of Bio-Rad
Laboratories, Inc.
http://instruct1.cit.cornell.edu/courses/biobm330/protlab/Gel_filtration.html